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Entering edit mode
5 weeks ago

Hello Biostars community,

I am trying to create a KO of my target gene. After editing my target gene in CRISPOR, I used the cDNA without my KO region into expasy translate to find out the possible outcome after translation.

Here we find the 6 different open reading frames in the forward and the reverse direction. One of my reading frames, is very similar to the target WT protein, which I also confirmed through BLAST.

How to identify if this particular ORF will be biologically relevant, meaning to ask if this ORF will indeed be detected as a coding sequence and translated by a eukaryotic cell system? Any tools available to check the likelihood of which ORF will be detected as the coding sequence region?

KO ORF KOZAK BLAST CDS • 125 views
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Entering edit mode
5 weeks ago

Best prove of existence is the blast approach you did, if that gene sequence seems to be conserved between species it's likely a true gene. A similar approach would be to check if you can find transcript (expression) evidence for it.

alternatively you could run an ORF finding software on it (TransDecoder? FrameD? ) and check if they pick this up (== they use some coding models to determine the likelihood of being a true gene).

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Entering edit mode

CodAn and borf are options also. The latter is available as a python module. OP could also check whether the translation contains the "right" domains (e.g., by using InterProScan).